Molecular marker for the early detection of malignant pleural mesothelioma and the methods of its expression analysis using blood and pleural effusion samples

a mesothelioma and pleural effusion technology, applied in the field of mesothelioma detection methods, can solve the problems of belated diagnosis, poor prognosis, and unfavorable prognosis of malignant pleural mesothelioma, and achieve the effects of low cost, high reliability, and simple structur

Inactive Publication Date: 2014-12-11
NAGOYA UNIVERSITY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For malignant pleural mesothelioma, treatments involving a combination of therapies of surgery, internal medicine and radiology have been carried out; however, malignant pleural mesothelioma has been still one of tumors accompanied by poor prognoses.
The long latency period, along with the lack of a screening method useful for early detection, results in belated diagnosis of the disease condition.
In advanced cases, the prognosis can be extremely unfavorable.
Inmost of such cases, the disease states advance rapidly, leading to a death within several years.
Accordingly, development of a novel screening method that allows early detection has been an urgent issue.
Early diagnoses of malignant pleural mesothelioma are extremely difficult, and the disease state already falls under an advanced stage in most cases when malignant pleural mesothelioma was found due to subjective symptoms such as chest pain, and respiratory distress resulting from pleural effusion.
Examples of a marker for use in diagnoses of malignant pleural mesothelioma are comprised of hyaluronic acid, CYFRA, CEA and the like in the pleural fluid; however, these involve significant problems such as necessities for collecting the pleural fluid as a sample, and inferior accuracy of the diagnosis.
Moreover, in order to make a reliable diagnosis, it is necessary to carry out a precise histopathological diagnosis using a biopsy specimen, and thus an extremely heavy psychological, physical and social burden is imposed.
Of them, prognosis of the sarcomatoid malignant pleural mesothelioma is extremely poor, without any effective diagnostic method.
Therefore, when malignant mesothelioma is diagnosed, in many cases, it has already been widely spread, and it is often impossible to carry out a radical surgery.
Accordingly, prognoses are extremely poor, with one and two year survival rate of about 50% and about 20%, respectively.
Therefore, X-ray examination has been considered to less useful as a means for screening.
For diagnoses of sarcomatoid pleural mesothelioma, cytological examination, pleura biopsy and image diagnosis have been proposed to be needed, and thus the diagnosis thereof has been extremely difficult (Nonpatent Document 2).
However, these cancers are completely different from mesothelioma in terms of disease conditions.
However, a relationship of mesothelioma with periostin expression and the presence in blood has not been known so far.

Method used

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  • Molecular marker for the early detection of malignant pleural mesothelioma and the methods of its expression analysis using blood and pleural effusion samples
  • Molecular marker for the early detection of malignant pleural mesothelioma and the methods of its expression analysis using blood and pleural effusion samples
  • Molecular marker for the early detection of malignant pleural mesothelioma and the methods of its expression analysis using blood and pleural effusion samples

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of Anti-Periostin Antibody

[0066](1) Structure of Human Periostin Protein

[0067]FIG. 1 shows a schematic view illustrating relation among positions of functional domains of a human periostin protein, the structure of each isoform, and the structure of the immunogen used for producing the periostin antibody of the present invention. In FIG. 1, EMI and Fasciclin 1 represent an EMILIN homologous domain and a fasciclin homologous domain, respectively. “iso1” to “iso4” represent isoforms 1 to 4 of human periostin, respectively. POSTN781 represents a recombinant protein obtained by fusing myc tag and his tag (represented by “mychis”) to a carboxyl terminal of a polypeptide having 781 amino acid residues of a human periostin protein isoform 3. POSTN630 represents a recombinant protein obtained by fusing myc tag and his tag to a carboxyl terminal of a polypeptide having 630 amino acid residues that are common to all isoforms of the human periostin protein covering four fasciclin do...

example 2

Measurement of Clinical Specimen

[0083](1) Reactivity to Clinical Specimen

[0084]With regard to the specimens from healthy individuals, an approval by Ethics Committee Organization of Medical & Biological Laboratories Co., Ltd.; MBL ethics review board (Project number: 022; Date of Approval: Mar. 27, 2007) was obtained and then specimens from healthy volunteers were solicited. Written consent for the measurement was obtained beforehand from each healthy individual who donated the specimen. With regard to specimens from mesothelioma patients, the aforementioned plasma specimens diluted 1,000 fold were used in place of human periostin purified recombinant proteins for the sandwich ELISA method explained in Example 1, section (7) which had been requested and obtained from BMR (Bio Medical Resources (integrated with Sera Care Life Sciences Milford, Mass., at the time of application)). Results of measurements in connection with the combinations of antibodies shown in Table 1 on the plasma ...

example 3

Measurement of Clinical Specimens

[0088](1) Measurement of Concentration of Periostin in Plasma Samples of Mesothelioma Patients and Healthy Individuals

[0089]Each 50 μL of the anti-human periostin monoclonal antibody #6-14-3 diluted to 10 μg / mL with a solution of 0.1 M NaHCO3, 0.1 M Na CO3 and 0.15% Proclin 150 (SUPELCO) was coated on a MaxiSorp 96-well plate (NUNC, Thermo Fisher Scientific K.K.), and left to stand at 4° C. overnight or at room temperature for 2 hours to allow the antibody to be immobilized. After the solution of the antibody was eliminated, each 150 μL of a blocking buffer (PBS supplemented with 1% BSA (Proliant, Veritas Corporation), 0.15% Proclin 150 and 5% sucrose) was dispensed, followed by leaving to stand at 4° C. overnight or at room temperature for 2 hours. After the blocking buffer solution was eliminated, using PBS supplemented with 1% BSA, 0.1% Tween20, 0.15% Proclin 150 and 50 μg / mL MAK-33 as a dilution liquid, a periostin standard substance (a series of...

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Abstract

The present invention provides a method for testing mesothelioma comprising a step of determining a concentration of a human periostin protein in at least one type of sample of blood or pleural fluid of a subject. In the step of determining the concentration of human periostin protein, an antibody directed against human periostin protein may be used. The present invention further provides a kit for diagnosing mesothelioma, said kit comprising an antibody directed against human periostin protein. In the kit for diagnosing mesothelioma, the antibody directed against a human periostin protein may be an antibody that binds to a polypeptide consisting of an amino acid sequence set out in SE ID NO: 2.

Description

TECHNICAL FIELD[0001]The present invention relates to a method for testing mesothelioma, and a kit for diagnosing mesothelioma. Specifically, the present invention relates to a method for testing mesothelioma, comprising a step of determining the concentration of human periostin in at least one type of samples of blood and pleural fluid, and a kit for diagnosing mesothelioma, comprising an antibody directed against a human periostin protein.BACKGROUND ART[0002]Mesothelioma has been known to be caused by inhalation of asbestos. Mesothelioma is classified into pleural mesothelioma, peritoneal mesothelioma and pericardial mesothelioma in accordance with the location where it develops. Mesothelioma most frequently occurs at pleura, and pleural mesothelioma accounts for 80 to 90% of the entirety of the mesothelioma episodes (Nonpatent Document 1). Benign pleural mesothelioma is referred to as fibrous mesothelioma, and more specifically, as a result of pneumonia, injury or the like follow...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/574C07K16/18
CPCC07K16/18G01N33/57423G01N33/57407C07K14/47
Inventor YANAGISAWA, KIYOSHITAKAHASHI, TAKASHIYOKOI, KOHEIHASEGAWA, YOSHINORIONO, KEN-ICHIROYAGI, KASUMIMASUDA, HITOMITAKEUCHI, TOSHIYUKI
Owner NAGOYA UNIVERSITY
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